CN110038523B - Preparation method of affinity chromatography medium for separating and purifying hexokinase - Google Patents
Preparation method of affinity chromatography medium for separating and purifying hexokinase Download PDFInfo
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Abstract
The invention provides a preparation method of an affinity chromatography medium for separating and purifying hexokinase, which comprises the following steps: s1, firstly, adopting agarose gel as a matrix, and preparing aldehyde agarose gel through the aldehydic reaction of sodium periodate solution; s2, coupling glucosamine serving as an affinity ligand to the aldehyde agarose gel prepared in the step S1; s3, reducing the coupling product prepared in the step S2 by adopting a sodium borohydride solution to prepare a stable affinity chromatography medium; s4, washing the affinity chromatography medium prepared in the step S3 with distilled water, draining, and storing in ethanol solution for later use. The preparation method provided by the invention has the advantages of low price of raw materials, easiness in obtaining, simple preparation process, stable and reliable purification method, easiness in control, low requirement on equipment, good application prospect and good economic and social benefits.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of an affinity chromatography medium for separating and purifying hexokinase.
Background
Hexokinase (Hexokinase) is a six-carbon sugar phosphorylase that uses hexose as a specific substrate, and plays an important role in the glycolysis process of organisms. Hexokinase is an allosteric enzyme, catalyzes glucose to change from a stable state to an active state, has a small Km value, strong affinity to substrates and weak specificity, and can act on various hexoses. By utilizing the action principle and characteristics of the enzyme, the enzyme is mainly used for clinical diagnosis reagents, particularly clinical detection of glucose in blood. Hexokinase is used as a key enzyme in the preparation of a diagnostic kit, and the stability of the hexokinase directly influences the stability of the diagnostic kit. Therefore, the preparation of hexokinase with high stability is the key to the preparation of a diagnostic kit with good stability. However, since hexokinase is derived from microbial fermentation broth, other biomacromolecules, especially proteases, existing in the fermentation broth can seriously affect the stability of hexokinase, so that the hexokinase is difficult to store for a long time. Therefore, the method has great significance for quickly and efficiently separating and purifying the hexokinase from the fermentation liquor and preparing the hexokinase with high stability.
The affinity chromatography is a liquid chromatography method for performing affinity adsorption on a target product by using an affinity adsorption medium coupled with an affinity ligand as a stationary phase so as to separate and purify the target product. Affinity chromatography has the characteristics of high selectivity, high activity recovery rate, high purity and the like, and is one of the most effective technologies for purifying biological macromolecules such as proteins and the like.
The affinity ligand is the core of the affinity chromatography medium and determines the efficiency of the affinity chromatography separation. Glucosamine belongs to hexose, can be used as a substrate of hexokinase, and has a certain affinity for hexokinase, and no report has been made so far on how to separate hexokinase by affinity chromatography using glucosamine as an affinity ligand.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides, in a first aspect, a method for preparing an affinity chromatography medium for isolating and purifying hexokinase, comprising the steps of:
s1, firstly, adopting agarose gel as a matrix, and preparing aldehyde agarose gel through the aldehydic reaction of sodium periodate solution;
s2, coupling glucosamine serving as an affinity ligand to the aldehyde agarose gel prepared in the step S1;
s3, reducing the coupling product prepared in the step S2 by adopting a sodium borohydride solution to prepare a stable affinity chromatography medium;
s4, washing the affinity chromatography medium prepared in the step S3 with distilled water, draining, and storing in ethanol solution for later use.
Wherein the agarose gel is selected from one or the combination of any several of agarose 6FF gel, agarose 4FF gel, agarose CL-6B gel, agarose CL-4B gel, agarose 6B gel and agarose 4B gel.
Wherein the mass ratio of the agarose gel to the sodium periodate is 10: 1-5.
Preferably, the mass ratio of the agarose gel to the sodium periodate is 10:2, 10:3 and 10: 4.
Wherein the mass ratio of the aldehyde agarose gel to the glucosamine is 10: 5-15.
Preferably, the mass ratio of the aldehyde agarose gel to the glucosamine is 10:6, 10:7, 10:8, 10:9, 10:10, 10:11, 10:12, 10:13, 10: 14.
Wherein the concentration of the ethanol solution is 10-25%.
Preferably, the concentration of the ethanol solution is 12%, 14%, 16%, 18%, 20%, 22%, 24%.
Wherein, in the step S1, the temperature of the hydroformylation reaction is 15-40 ℃, the time is 0.5-3h, and the rotation speed is 100-300 rpm.
Wherein, in the step S2, the temperature of the coupling reaction is 25-40 ℃, the time is 8-24h, and the rotation speed is 100-300 rpm.
Wherein, in the step S3, the temperature of the reduction reaction is 2-6 ℃ and the time is 1-5 h.
Wherein, in the step S4, the temperature for storing the affinity chromatography medium is 2-6 ℃.
In a second aspect, the present invention provides a method for isolating and purifying hexokinase, comprising the steps of:
s1, centrifuging the fermentation liquor containing hexokinase to remove thalli to obtain completely clarified fermentation liquor;
s2, incubating the sample obtained in the step S1 by using the affinity chromatography medium prepared by the method provided by the first aspect of the invention;
s3, eluting the hexokinase adsorbed by the affinity chromatography medium in the step S2.
The invention has the beneficial effects that:
the preparation method of the affinity chromatography medium for separating and purifying hexokinase provided by the invention has the following advantages:
1. the glucosamine for preparing the affinity ligand is easy to obtain and has low price;
2. the preparation process has the characteristics of simple operation, stable and reliable purification method, easy control and low requirement on equipment;
3. provides a new preparation process for separating and purifying the affinity ligand of hexokinase, and has good application prospect and good economic and social benefits.
Detailed Description
The following is a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements are also considered to be within the scope of the present invention.
Example 1
The invention provides a preparation method of an affinity chromatography medium for separating and purifying chitosanase, which comprises the following steps:
s1, firstly, placing 5g of washed and drained agarose 6FF gel into a 250mL triangular flask, adding 50mL of sodium periodate solution with the concentration of 0.01g/mL, placing the mixture into a constant-temperature shaking incubator, controlling the temperature to be 25 ℃ and the rotating speed to be 150rpm, and carrying out an hydroformylation reaction for 1h to prepare hydroformylation agarose 6FF gel;
s2, taking 1g of the aldehyde agarose gel prepared in the step S1, placing the aldehyde agarose gel in a sand core funnel, washing the aldehyde agarose gel with distilled water, draining the washed aldehyde agarose gel, transferring the aldehyde agarose gel into a 250mL triangular flask, adding 50mL of glucosamine solution with the concentration of 0.01g/mL, placing the product in a constant-temperature shaking incubator, controlling the temperature to be 37 ℃ and the rotating speed to be 150rpm, and carrying out coupling reaction for 12 hours;
s3, transferring the glucosamine-coupled agarose gel prepared in the step S2 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, transferring the sand core funnel into a 250mL triangular flask, adding 50mL of 0.01g/mL sodium borohydride solution, placing the flask in a refrigerator at the temperature of 4 ℃, and carrying out reduction reaction for 2 hours to prepare a stable affinity chromatography medium;
s4, transferring the affinity chromatography medium prepared in the step S3 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, and storing the sand core funnel in 20% ethanol solution at 4 ℃ for later use.
The method for separating and purifying hexokinase by using the affinity chromatography medium prepared in example 1 comprises the following steps:
s1, firstly, 5ml of fermentation liquor containing hexokinase is centrifuged at 6000rpm for 15min to remove thalli, and completely clarified fermentation liquor is obtained;
s2, loading 10ml of the affinity chromatography medium prepared in the example 1 into a chromatographic column (phi 1.0 cm multiplied by 10 cm), and balancing with 100ml of distilled water;
s3, adding the completely clarified fermentation liquor obtained in the step S1 into a chromatographic column, incubating for 10 min, eluting the hybrid protein by using a 1% sodium chloride solution, setting the flow rate to be 30mL/h, eluting for 2h, eluting the hexokinase by using a 1% glucose solution, and eluting for 1h at a flow rate of 20 mL/h.
The agarose gel of example 1 can also be selected from one or more of agarose 6FF gel, agarose 4FF gel, agarose CL-6B gel, agarose CL-4B gel, agarose 6B gel and agarose 4B gel.
In order to verify the separation and purification effects of the affinity chromatography media prepared from different agarose gels on hexokinase, the first set of comparative experiments was set by changing the components and the ratio of the agarose gel according to table one, with example 1 as a reference, and with the experimental conditions and parameters being kept constant.
TABLE-Effect of different compositions of agarose gel on the isolation and purification of hexokinase
| Comparative test | The components and the proportion of the agarose gel | Separation and purification fold of hexokinase |
| Comparative experiment 1 | Agarose 6FF gel 5g | 12.6 |
| Comparative experiment 2 | Agarose 4FF gel 5g | 13.8 |
| Comparative experiment 3 | Agarose CL-6B gel 5g | 12.5 |
| Comparative experiment 4 | Agarose CL-4B gel 5g | 12.3 |
| Comparative experiment 5 | Agarose 6B gel 5g | 13.2 |
| Comparative test 6 | Agarose 4B gel 5g | 12.2 |
| Comparative experiment 7 | Agarose 6FF gel 3g, agarose CL-6B gel 2g | 13.8 |
| Comparative experiment 8 | Agarose 6FF gel 2g, agarose CL-6B gel 1g, agarose 4B gel 2g | 16.2 |
| Comparative test 9 | Agarose 4FF gel 1g, agarose CL-6B gel 2g, agarose 6B gel 2g | 14.9 |
| Comparative experiment 10 | Agarose 6FF gel 1g, agarose 4FF gel 0.5g, agarose CL-6B gel 1g, agarose CL-4B gel 1g, agarose 6B gel 0.5, agarose 4B gel 1g | 15.3 |
As can be seen from the comparative tests, the agarose gel comprises any one or a combination of several of the agarose gel components (refer to comparative tests 1-10), and the prepared affinity chromatography medium can separate and purify the chitosanase; when the agarose gel comprises two or more than two components (refer to a comparative experiment 7-10), the separation and purification effects of the chitosanase are obviously better than those of the comparative experiments 1-6; accordingly, the agarose gel is preferably two or more of the above agarose gels.
Example 2
The invention provides a preparation method of an affinity chromatography medium for separating and purifying hexokinase, which comprises the following steps:
s1, firstly, putting 2g of washed and drained agarose 6FF gel, 1g of agarose CL-6B gel and 2g of agarose 4B gel into a 250mL triangular flask, adding 50mL of sodium periodate solution with the concentration of 0.03g/mL, putting the mixture into a constant-temperature shaking incubator, controlling the temperature to be 25 ℃ and the rotating speed to be 150rpm, and carrying out an hydroformylation reaction for 1 hour to prepare the hydroformylation agarose 6FF gel;
s2, taking 1g of the aldehyde agarose gel prepared in the step S1, placing the aldehyde agarose gel in a sand core funnel, washing the aldehyde agarose gel with distilled water, draining the washed aldehyde agarose gel, transferring the aldehyde agarose gel into a 250mL triangular flask, adding 50mL of glucosamine solution with the concentration of 1g/mL, placing the product in a constant-temperature shaking incubator, controlling the temperature to be 37 ℃ and the rotating speed to be 150rpm, and carrying out coupling reaction for 12 hours;
s3, transferring the glucosamine-coupled agarose gel prepared in the step S2 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, transferring the sand core funnel into a 250mL triangular flask, adding 50mL of 0.01g/mL sodium borohydride solution, placing the flask in a refrigerator at the temperature of 4 ℃, and carrying out reduction reaction for 2 hours to prepare a stable affinity chromatography medium;
s4, transferring the affinity chromatography medium prepared in the step S3 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, and storing the sand core funnel in 20% ethanol solution at 4 ℃ for later use.
The method for separating and purifying hexokinase by using the affinity chromatography medium prepared in example 2 comprises the following steps:
s1, firstly, 5ml of fermentation liquor containing hexokinase is centrifuged at 6000rpm for 15min to remove thalli, and completely clarified fermentation liquor is obtained;
s2, loading 10ml of the affinity chromatography medium prepared in the example 1 into a chromatographic column (phi 1.0 cm multiplied by 10 cm), and balancing with 100ml of distilled water;
s3, adding the completely clarified fermentation liquor obtained in the step S1 into a chromatographic column, incubating for 10 min, eluting the hybrid protein by using a 1% sodium chloride solution, setting the flow rate to be 30mL/h, eluting for 2h, eluting the hexokinase by using a 1% glucose solution, and eluting for 1h at a flow rate of 20 mL/h.
In order to verify the separation and purification effects of affinity chromatography media prepared from agarose gel and sodium periodate with different mass ratios on hexokinase, a second set of comparative experiments was set by changing the concentration of sodium periodate by taking example 2 as reference and controlling the experimental conditions and parameters to be unchanged, as shown in table two.
Influence of agarose gel and sodium periodate with different mass ratios on hexokinase separation and purification effect
| Comparative test | Quality (g) of sodium periodate | Mass ratio of agarose gel to sodium periodate | Separation and purification fold of hexokinase |
| Comparative experiment 1 | 0.1 | 10:0.2 | 3.7 |
| Comparative experiment 2 | 0.25 | 10:0.5 | 8.6 |
| Comparative experiment 3 | 0.5 | 10:1 | 16.2 |
| Comparative experiment 4 | 1.0 | 10:2 | 18.9 |
| Comparative experiment 5 | 1.5 | 10:3 | 20.5 |
| Comparative test 6 | 2.0 | 10:4 | 13.2 |
| Comparative experiment 7 | 2.5 | 10:5 | 11.5 |
| Comparative experiment 8 | 3.0 | 10:6 | 6.3 |
As can be seen from the above comparative experiments, the effect of isolating and purifying hexokinase by the prepared affinity chromatography medium is optimal when the mass ratio of the agarose gel to the sodium periodate is 10:1-5, and therefore the mass ratio of the agarose gel to the sodium periodate is preferably 10: 1-5.
Example 3
The invention provides a preparation method of an affinity chromatography medium for separating and purifying hexokinase, which comprises the following steps:
s1, firstly, putting 2g of washed and drained agarose 6FF gel, 1g of agarose CL-6B gel and 2g of agarose 4B gel into a 250mL triangular flask, adding 50mL of sodium periodate solution with the concentration of 0.03g/mL, putting the mixture into a constant-temperature shaking incubator, controlling the temperature to be 20 ℃ and the rotating speed to be 250rpm, and carrying out an hydroformylation reaction for 2 hours to prepare the hydroformylation agarose 6FF gel;
s2, taking 1g of the aldehyde agarose gel prepared in the step S1, placing the aldehyde agarose gel in a sand core funnel, washing the aldehyde agarose gel with distilled water, draining the washed aldehyde agarose gel, transferring the aldehyde agarose gel into a 250mL triangular flask, adding 50mL of glucosamine solution with the concentration of 1g/mL, placing the product in a constant-temperature shaking incubator, controlling the temperature to be 30 ℃ and the rotating speed to be 200rpm, and carrying out coupling reaction for 20 hours;
s3, transferring the glucosamine-coupled agarose gel prepared in the step S2 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, transferring the sand core funnel into a 250mL triangular flask, adding 50mL of 0.01g/mL sodium borohydride solution, placing the flask in a refrigerator at the temperature of 5 ℃, and performing reduction reaction for 3 hours to prepare a stable affinity chromatography medium;
s4, transferring the affinity chromatography medium prepared in the step S3 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, and storing the sand core funnel in 15% ethanol solution at 3 ℃ for later use.
The method for separating and purifying hexokinase by using the affinity chromatography medium prepared in example 3 comprises the following steps:
s1, firstly, 5ml of fermentation liquor containing hexokinase is centrifuged at 6000rpm for 15min to remove thalli, and completely clarified fermentation liquor is obtained;
s2, loading 10ml of the affinity chromatography medium prepared in the example 1 into a chromatographic column (phi 1.0 cm multiplied by 10 cm), and balancing with 100ml of distilled water;
s3, adding the completely clarified fermentation liquor obtained in the step S1 into a chromatographic column, incubating for 10 min, eluting the hybrid protein by using a 1% sodium chloride solution, setting the flow rate to be 30mL/h, eluting for 2h, eluting the hexokinase by using a 1% glucose solution, and eluting for 1h at a flow rate of 20 mL/h.
In order to verify the separation and purification effects of the affinity chromatography media prepared from the aldehyde sepharose gel and the glucosamine with different mass ratios on hexokinase, the experiment conditions and parameters were controlled to be unchanged by taking the example 2 as reference, and a third set of comparative experiments were set by changing the concentration of the glucosamine, as shown in table three.
Influence of aldehyde agarose gel and glucosamine with different mass ratios on hexokinase separation and purification effect
| Comparative test | Quality of glucosamine (g) | Mass ratio of aldehyde agarose gel to glucosamine | Separation and purification fold of hexokinase |
| Comparative experiment 1 | 0.1 | 10:1 | 9.8 |
| Comparative experiment 2 | 0.3 | 10:3 | 10.2 |
| Comparative experiment 3 | 0.5 | 10:5 | 16.2 |
| Comparative experiment 4 | 0.7 | 10:7 | 13.7 |
| Comparative experiment 5 | 0.9 | 10:9 | 12.9 |
| Comparative test 6 | 1.1 | 10:10 | 10.2 |
| Comparative experiment 7 | 1.3 | 10:13 | 8.9 |
| Comparative experiment 8 | 1.5 | 10:15 | 6.5 |
| Comparative test 9 | 1.7 | 10:17 | 6.3 |
| Comparative experiment 10 | 2.0 | 10:20 | 5.9 |
As can be seen from the comparative tests, when the mass ratio of the aldehyde agarose gel to the glucosamine is 10:3-10, the prepared affinity chromatography medium has the best effect of separating and purifying the hexokinase, and therefore, the mass ratio of the aldehyde agarose gel to the glucosamine is preferably 10: 3-10.
Example 4
The invention provides a preparation method of an affinity chromatography medium for separating and purifying hexokinase, which comprises the following steps:
s1, firstly, putting 2g of washed and drained agarose 6FF gel, 1g of agarose CL-6B gel and 2g of agarose 4B gel into a 250mL triangular flask, adding 50mL of sodium periodate solution with the concentration of 0.03g/mL, putting the mixture into a constant-temperature shaking incubator, controlling the temperature to be 30 ℃ and the rotating speed to be 200rpm, and carrying out an hydroformylation reaction for 1.5 hours to prepare the hydroformylation agarose 6FF gel;
s2, taking 1g of the aldehyde agarose gel prepared in the step S1, placing the aldehyde agarose gel in a sand core funnel, washing the aldehyde agarose gel with distilled water, draining the washed aldehyde agarose gel, transferring the aldehyde agarose gel into a 250mL triangular flask, adding 50mL of glucosamine solution with the concentration of 1g/mL, placing the product in a constant-temperature shaking incubator, controlling the temperature to be 35 ℃ and the rotating speed to be 300rpm, and carrying out coupling reaction for 10 hours;
s3, transferring the glucosamine-coupled agarose gel prepared in the step S2 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, transferring the sand core funnel into a 250mL triangular flask, adding 50mL of 0.01g/mL sodium borohydride solution, placing the flask in a refrigerator at the temperature of 5 ℃, and performing reduction reaction for 4 hours to prepare a stable affinity chromatography medium;
s4, transferring the affinity chromatography medium prepared in the step S3 into a sand core funnel, washing the sand core funnel with distilled water, draining the sand core funnel, and storing the sand core funnel in 25% ethanol solution at the temperature of 5 ℃ for later use.
The method for separating and purifying hexokinase by using the affinity chromatography medium prepared in example 4 comprises the following steps:
s1, firstly, 5ml of fermentation liquor containing hexokinase is centrifuged at 6000rpm for 15min to remove thalli, and completely clarified fermentation liquor is obtained;
s2, loading 10ml of the affinity chromatography medium prepared in the example 1 into a chromatographic column (phi 1.0 cm multiplied by 10 cm), and balancing with 100ml of distilled water;
s3, adding the completely clarified fermentation liquor obtained in the step S1 into a chromatographic column, incubating for 10 min, eluting the hybrid protein by using a 1% sodium chloride solution, setting the flow rate to be 30mL/h, eluting for 2h, eluting the hexokinase by using a 1% glucose solution, and eluting for 1h at a flow rate of 20 mL/h.
To verify the separation and purification effect of glucose solutions with different concentrations on hexokinase, using example 4 as a reference, the experimental conditions and parameters were kept unchanged, and a fourth set of comparative experiments were set up by varying the glucose concentration, as shown in table four.
TABLE four Effect of elution with different glucose concentrations on the isolation and purification effects of hexokinase
| Comparative test | Glucose concentration (%) | Separation and purification fold of hexokinase |
| Comparative experiment 1 | 0.5 | 7.8 |
| Comparative experiment 2 | 0.6 | 9.2 |
| Comparative experiment 3 | 0.7 | 11.6 |
| Comparative experiment 4 | 0.8 | 12.7 |
| Comparative experiment 5 | 0.9 | 14.9 |
| Comparative test 6 | 1.0 | 16.2 |
| Comparative experiment 7 | 1.1 | 17.6 |
| Comparative experiment 8 | 1.2 | 15.3 |
| Comparative test 9 | 1.3 | 15.1 |
| Comparative experiment 10 | 1.4 | 14.2 |
As can be seen from the above comparative experiments, the affinity chromatography medium prepared has the best effect of isolating and purifying hexokinase when the glucose concentration is 0.9-1.4%, and therefore the glucose concentration is preferably 0.9-1.4%.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the spirit of the present invention, and these changes and modifications are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. A preparation method of an affinity chromatography medium for separating and purifying hexokinase is characterized by comprising the following steps:
s1, firstly, adopting agarose gel as a matrix, and preparing and obtaining aldehyde agarose gel through the aldehyde reaction of a sodium periodate solution, wherein the mass ratio of the agarose gel to the sodium periodate is 10: 1-5;
s2, coupling glucosamine serving as an affinity ligand to the aldehyde agarose gel prepared in the step S1;
s3, reducing the coupling product prepared in the step S2 by adopting a sodium borohydride solution to prepare a stable affinity chromatography medium;
s4, washing the affinity chromatography medium prepared in the step S3 with distilled water, draining, and storing in ethanol solution for later use.
2. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to claim 1, wherein: the agarose gel is selected from one or the combination of any more of agarose 6FF gel, agarose 4FF gel, agarose CL-6B gel, agarose CL-4B gel, agarose 6B gel and agarose 4B gel.
3. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to claim 1, wherein: the mass ratio of the aldehyde agarose gel to the glucosamine is 10: 5-15.
4. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to claim 1, wherein: the concentration of the ethanol solution is 10-25%.
5. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to any one of claims 1-4, wherein: in the step S1, the temperature of the hydroformylation reaction is 15-40 ℃, the time is 0.5-3h, and the rotation speed is 100-300 rpm.
6. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to any one of claims 1-4, wherein: in the step S2, the temperature of the coupling reaction is 25-40 ℃, the time is 8-24h, and the rotation speed is 100-300 rpm.
7. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to any one of claims 1-4, wherein: in the step S3, the temperature of the reduction reaction is 2-6 ℃ and the time is 1-5 h.
8. The method for preparing an affinity chromatography medium for isolating and purifying hexokinase according to any one of claims 1-4, wherein: in step S4, the temperature for storing the affinity chromatography medium is 2-6 ℃.
9. A method for separating and purifying hexokinase, comprising the steps of:
s1, centrifuging the fermentation liquor containing hexokinase to remove thalli to obtain completely clarified fermentation liquor;
s2 incubating the fermentation broth obtained in step S1) with the affinity chromatography medium prepared according to any one of claims 1 to 8;
s3, eluting the hexokinase adsorbed by the affinity chromatography medium in the step S2.
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| CN105944686A (en) * | 2016-05-18 | 2016-09-21 | 绿麒（厦门）海洋生物科技有限公司 | Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres |
| CN106479992A (en) * | 2016-11-24 | 2017-03-08 | 河北省微生物研究所 | The preparation method of diagnosis hexokinase |
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| US4666604A (en) * | 1984-09-26 | 1987-05-19 | Centre National De La Recherche Scientifique | New elution complex more especially for affinity chromatography and affinity precipitation |
| CN101591441A (en) * | 2009-02-23 | 2009-12-02 | 协和生物制药（天津）有限公司 | The method that a kind of hyaluronic acid is connected with sepharose |
| CN103586008A (en) * | 2013-10-24 | 2014-02-19 | 中国科学院过程工程研究所 | Affinity chromatography medium and preparation method and application thereof |
| CN105944686A (en) * | 2016-05-18 | 2016-09-21 | 绿麒（厦门）海洋生物科技有限公司 | Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres |
| CN106479992A (en) * | 2016-11-24 | 2017-03-08 | 河北省微生物研究所 | The preparation method of diagnosis hexokinase |
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